The mammalian collecting duct system by virtue of its downstream position in the kidney, its heterogenous cell population, and its role as a target tissue for major hormones such as arginine vasopressin and aldosterone plays a crucial role in the final composition of the urine. One of the more recently characterized portions of the collecting duct system is the outer medullary collecting ducts from the inner tripe of the outer medulla (OMCDi). This nephron segment specializes in net H+ secretion. The OMCDi accomplishes H+ secretion via an apical cell membrane H+ translocating ATPase which operates in series with a basolateral cell membrane Cl/HCO3 exchanger. Also, there is evidence for a major Cl- conductance channel on the basolateral membrane. Although there is evidence that aldosterone directly affects net H+ secretion by this nephron segment in vitro, very little else is know about the regulation of the acidification process in the OMCDi. The studies proposed in this grant are designed to provide important additional information. Three specific hypotheses will be tested: 1) A basolateral cell membrane Na+/H+ exchanger exists in cells of the OMCDi and contributes importantly to regulation of intracellular pH and possible to regulation of net acidification; 2) During in vivo K+ depletion there is a progressive alteration in the transport function of the OMCDi cells involving a phenotypic shift in the cells to express new or greater amounts of certain transporters responsible for the adaptive changes in OMCDi function as well as changes in cell size resulting in hypertrophy; 3) Short term regulation of OMCDi transport is accomplished by a number of hormones and autocoids which utilize two major signal transduction pathway, the cyclic AMP pathway and the phosphatidylinositol turnover pathway. These two signal transduction pathways, predominately affect the basolateral cell membrane transporters in opposite ways. To test these specific aims we will utilize the technique of in vitro microperfusion of OMCDi with measurement of net HCO3- flux as well as determination of intracellular ion activities utilizing a number of different ion sensitive fluorescent dyes (pH, Cl+, and Ca++). In addition some studies will utilize the technique of single cell puncture with microelectrodes to measure intracellular voltage. These studies will characterize certain important features of the apical and basolateral cell membrane transporters and their modification in K+ depletion as well as their regulation by a number of potentially important modulators.